The Laboratory Working Group of the NKDEP suggests that HPLC provides a fairly sensitive and analytically specific method for measuring SCr [3
]. Sample deproteinization improves the specificity of creatinine measurement by HPLC by removing many protein-bound endogenous and exogenous compounds without altering the quantification of creatinine [3
]. Data from CKiD subjects indicate that the Siemens creatinine enzymatic assay used by the Central Biochemistry Laboratory compares favorably with HPLC. This assay is reliable and more than adequate for the level of SCr encountered in children with moderate to severe CKD. With statistically significant numerical differences of only 7%, the enzymatic SCr values were slightly higher than those determined by HPLC, but the differences in the methods were within the manufacturers’ stated variability. Importantly, when the bias was examined along the entire range of SCr, there was no significant change. When a separate group of SCr samples from apparently normal infants and children with low SCr levels was examined, however, there was not a high agreement between the two assays, and the Siemens enzymatic assay had a negative bias against the HPLC assay by 18%.
The poor agreement at the low range of SCr led us to examine the accuracy at the low concentration ranges associated with pediatric patients. Since NIST does not have a SRM at this low concentration (Level I was at 0.6355 mg/dl), the only recourse, though less than ideal, was to dilute the NIST SRM Level 1 to achieve a target value of approximately 0.32 mg/dl. Even if the exact value were uncertain due to introduction of errors associated with the dilution step and the use of BSA as the diluent, it was still possible to obtain an assessment of accuracy and precision of the commercial enzymatic assays. HPLC and Roche enzymatic assays gave means of 0.31 mg/dl, which agreed very well with the estimated target value of 0.32 mg/dl. Siemens enzymatic method mean was 0.36 mg/dl, overestimating the target value by 16%. More importantly, the imprecision of the Siemens enzymatic assay was concerning, with a CV of 16% compared with 2.3% for the Roche and 1.6–2% for the HPLC assay.
Difficulties in measuring SCr at the low range have been previously encountered using the Jaffe colorimetric reaction [9
], and efforts to improve precision at this level included initial dialysis steps, adsorption phases, and increasing signal to noise ratios (reviewed in [1
]). NIST provides a “low-level” standard of 0.6355 mg/dl, which is twice the expected value of SCr for a healthy infant or toddler. Although HPLC assay linearity ranges extends to 0.15 mg/dl, accurate determination at the critical pediatric SCr range of 0.3 mg/dl is not possible due to the lack of a NIST SRM at that concentration range. Our resort to using a low “pediatric” standard prepared by dilution of the low SRM (Level I) made the HPLC method not commutable to an IDMS reference method due to the error introduced by the dilution process and the use of creatinine-free BSA instead of human serum as the diluent [10
]. Nevertheless, our data showed that one of the two commercial assays for SCr had good agreement with HPLC, whereas the other had a positive bias of 13%, and was 19% higher than HPLC values, a bias that could have clinically significant consequences.
The use of IDMS-based SRMs in the pediatric range should bring accuracy to the measurement of low SCr levels (achieving trueness in creatinine results), eliminate methodological bias among assay manufacturers, and ultimately standardize the creatinine measurement. Thus, accurate knowledge of normal SCr values could be assured prior to the use of nephrotoxic agents, chemotherapy, or total body irradiation.
Whereas the availability of SRM at low SCr levels will help improve accuracy, assay manufacturers should also focus on improving the precision of their assays at low SCr levels. Our data showed that one commonly used SCr assay had a CV of 16% at 0.32 mg/dl. Whereas the low SCr values of infants and young children are seemingly reassuring, a true increase of SCr from 0.3 to 0.4 with acute pyelonephritis is of major clinical significance.
In summary, at the SCr levels encountered in CKiD study patients (median 1.4 mg/dl), the Central Biochemistry Laboratory Siemens assay yielded results comparable with the HPLC assay, which was commutable to IDMS for the concentration range bracketed by the two levels of serum SRMs (Level I = 0.6355 mg/dl, Level II = 5.287 mg/d). We extended this study to investigate whether the Siemens assay could accurately measure low SCr levels associated with normal infants and children. The results proved that it could not. We showed that another commonly used analyzer (Roche) was comparable with HPLC in accuracy and precision for the diluted low standard.
Therefore, we suggest that physicians ordering SCr on infants and children be aware of the accuracy and reproducibility of the creatinine assay used in the clinical laboratories of their choice, especially for evaluating changes in low SCr values. Further, we suggest that NIST develop a human “pediatric” SCr standard reference material for use by assay manufacturers to improve the accuracy and precision of their assays at the low SCr expected in most pediatric patients. In the absence of such development, the ability of the physician to monitor the progression of mild CKD in infants and children and safely prescribe therapies in which the dose is correlated to kidney function would be compromised.